FEMS MicrobiologyLetters 20 (1983) 395-399
Published by Elsevier
395
Changes in antibiotic sensitivity and cell surface hydrophobicity in
Escherichia coli injured by heating, freezing, drying or gamma
radiation
(Gram-negative outer membrane; antibiotics; sublethal injury)
B.M. M a c k e y
ARC Meat Research Institute, Langford, Bristol BS18 7D Y, U.K.
Received 8 August 1983
Accepted 12 August 1983
1. SUMMARY
Escherichia coli cells exposed to mild heating,
freezing and thawing, drying or y-radiation were
sensitised to hydrophobic antibiotics and sodium
deoxycholate but not to small hydrophilic antibiotics. These stress treatments also caused increases
in cell surface hydrophobicity broadly reflecting
the degree of sensitivity to hydrophobic antibiotics.
2. I N T R O D U C T I O N
The outer membrane of enteric Gram-negative
bacteria acts as a permeability barrier to dyes,
detergents and certain antibiotics, protecting
bacteria from their inhibitory effects [1]. These
substances are often incorporated in selective
media used for isolating E. coli, salmonellae and
other enterobacteria from food. However, heating,
freezing, drying and other methods of preserving
food cause damage to the outer membrane resulting in loss of ability to grow on certain selective
media with a consequent likelihood of underestimating viable numbers in food. Although the practical implications of membrane damage are well
appreciated [2], little is known about the nature of
that damage and whether food preservation treatments differ in their effects.
By contrast, loss of the barrier properties of the
outer membrane has been intensively studied in
'deep rough' mutants of E. coli and Salmonella
having specific structural defects in the lipopolysaccharide (LPS) component of the membrane and
also in cells treated with EDTA [3,4]. In both cases
there is an increase in sensitivity to hydrophobic,
but not hydrophilic, substances believed to be due
to structural changes resulting in the formation of
hydrophobic regions on the previously hydrophilic
membrane surface [1].
To test whether physical damage has similar
effects, we have examined the sensitivity of frozen
and thawed, heated, dried and y-irradiated E. coli
cells to a range of hydrophilic and hydrophobic
antibiotics and measured changes in cell surface
hydrophobicity caused by the damage treatments.
3. MATERIALS AND M E T H O D S
3.1. Organism, growth conditions and media
E. coli K-12 strain C5 was grown at 37°C in
tryptone soya broth supplemented with 0.3% ( w / v )
0378-1097/83/$03.00 © 1983 Federation of European MicrobiologicalSocieties
396
yeast extract [5]. Viable numbers were estimated
on tryptone soya agar supplemented with 0.1%
( w / v ) sodium pyruvate (TSAP) to prevent inhibition of injured cells by peroxides in the medium
[5]. Filter-sterilised solutions of antibiotics were
added to molten TSAP as indicated.
3.2. Injury treatments
Injury treatments have been described in detail
previously [6]. Cells in the late l o g / e a r l y stationary phase (A680 = 1.0) were harvested by
centrifugation or filtration, washed and resuspended in 0.1 M potassium phosphate buffer p H
6.0 or 0.85% ( w / v ) sodium chloride. Washed cells
in phosphate buffer were subjected to one of the
following treatments: heating at 48°C for 30 or 60
min; irradiation from a 6°Co source to dose levels
of 120 or 240 k G y ; drying in albumin at 43°C.
Cells in saline suspension were frozen to - 1 0 ° C
for 20-22 h then thawed at 37°C. E D T A treatment was as described by Voll and Leive [7].
biotic. Plates were incubated at 37°C to constant
colony count and the concentrations of antibiotic
inhibiting 50% of cells (ICs0) determined. In the
screening method reported in Table 2, sensitivity is
expressed as the recovery on TSAP containing a
single concentration of antibiotic relative to the
recovery on TSAP alone. The concentration of
antibiotic chosen, had no effect on uninjured cells.
3.4. Hydrophobicity
Cell surface hydrophobicity was determined by
adherance to hydrocarbon using the o c t a n e / b u f f e r
two-phase system of Rosenberg et al. [8]. Increases
in hydrophobicity result in a decrease in cell concentration in the aqueous phase.
4. RESULTS
4.1. Changes in antibiotic sensitivity caused by freezing and thawing
3.3. Antibiotic sensitivity
Suitably diluted cell suspensions were spread on
TSAP containing graded concentrations of anti-
Freezing and thawing had little effect on the
sensitivity of E. coli to small, hydrophilic antibiotics (defined by Nikaido [3] as having an o c t a n o l /
Table 1
The effect of freezing and thawing on antibiotic resistance of Escherichia coli
Antibiotic
Cycloserine
Penicillin G
Ampicillin
Carbenicillin
Methicillin
Neomycin
Vancomycin
Polymyxin B
Bacitracin
Novobiocin
Erythromycin
Chlortetracycline
Nalidixic acid
Chloramphenicol
M~
102
334
349
378
379
615
c. 3300
c. 1200
1411
613
734
479
232
323
Partition ~
coefficient
< 0.01
0.02
< 0.01
< 0.01
0.01
< 0.01
< 0.01
< 0.05
0.21
> 20
0.79
0.31
3.16
12.4
Efficacy
ratio
IC5o
Unfrozen
Frozen/
thawed
16
21
2.5
3.9
> 2000
1.5
148
0.75
1 800
55
70
8
1.2
9
18
14
2.5
3.0
> 2000
1.25
1.1
0.28
49
0.2
3
1.4
0.58
4.5
0.9
1.5
1.0
1.3
1.2
135
2.7
37
275
23
5.7
2.1
2.0
a Partition coefficients, determined in octanol/0.05 M sodium phosphate buffer, pH 7, were taken from [3] and [9].
b ICs0 is the concentration of antibiotic (/~g/ml) required to inhibit 50% of organisms (see METHODS).
397
Table 2
The effect of antibiotics and sodium deoxycholate on the recovery of sublethally injured Escherichia coli
Treatment
% survival"
% surviving population inhibited by the test compound b
Sodium
deoxycholate
750/~g/ml
Sodium
deoxycholate
100/.t g / m l
Penicillin G
3 #g/ml
Vancomycin
20 ~tg/ml
Novobiocin
3 ~g/ml
Bacitracin
500 ~tg/ml
4
4
11
6
0
22
9
29 c
4l e
Heat
100
26
16
3
9
37 ~
0
0
2
0
10
0
Irradiation
100
8
0.5
19
44
77 e
4
1
10
0
3
5
3
19
61 e
2
11
33 a
16
31
56 d
Drying
100
0.01
24
> 95 e
1
66 e
0
0
8
62 e
4
0
19
58 ~
Freeze/thaw
100
7
12
86 ¢
8
50 e
7
13
12
62 ~
4
58 e
17
76 e
EDTA
ND
> 99e
> 99 ¢
2
65 e
96 e
98 e
"
Survival is expressed as the percentage of the original population able to form colonies on TSAP.
b
Sensitivity was determined by plating on TSAP with and without the test compound. See METHODS.
cd¢ Survivors were significantly more sensitive to the test compound than unstressed cells with P < 0.05, 0.01, 0.001, respectively.
Each tabulated value is the mean of five separate experiments. Data were subjected to analysis of variance.
ND, not determined.
water partition coefficient < 0.02), but sensitised
cells to vancomycin, a large hydrophilic molecule
(Table 1).
Hydrophobic antibiotics (partition coefficient
> 0.07) were all more inhibitory to frozen than
unfrozen cells, the increase in sensitivity, measured
as the change in IC50, varying from 2-fold to more
than 200-fold (Table 1).
Frozen
EDTA
Irradiated
4.2. Comparison of the effects of different treatments
0.3
All treatments resulted in outer membrane
damage, but the pattern of sensitivity to deoxycholate and antibiotics varied (Table 2). Dried,
frozen and EDTA-treated cells were more sensitive
to both high and low concentrations of deoxycholate than heated or irradiated cells. Frozen, irradiated and EDTA-treated cells were sensitised to
vancomycin, novobiocin and bacitracin, dried cells
to vancomycin and bacitracin and heated cells to
bacitracin only.
0.6
0.3
0.6
0.3
0.6
0.3
0.6
0crane 0n0
Fig. l. The effect of different stress treatments on cell surface
hydrophobicity of Escherichia coli. Cell surface hydrophobicity
was measured by adherence to liquid hydrocarbon [8]. Octane
was added to aqueous suspensions of cells and, after mixing
and allowing to stand, the absorbance of the aqueous phase
measured at 400 nm: closed circles, untreated cells; open
circles, frozen and thawed cells; open triangles, EDTA-treated
ceils; cells incubated in phosphate buffer, pH 6.0 for 60 min at
37°C (open squares) or 48°C (closed squares); open hexagons,
irradiated cells. A decrease in absorbance of the aqueous phase
indicates an increase in cell surface hydrophobicity.
398
4.3. Cell surface hydrophobicity
Large increases in hydrophobicity occurred in
frozen and EDTA-treated cells with smaller
increases in those heated or irradiated. Dried cells
were not tested owing to problems of obtaining
homogeneous suspensions after removal of albumin.
5. DISCUSSION
The barrier properties of the Gram-negative
outer membrane are believed to be due to its
unusual asymmetric structure [1]. The inner layer
of the membrane has a typical phospholipid/
protein composition whereas the outer layer consists predominantly of LPS and protein with little
exposed phospholipid. Hydrophobic molecules
penetrate this outer layer with difficulty because
they cannot pass through the highly charged hydrophilic regions of the LPS or the hydrophilic
exposed regions of the proteins. Small hydrophilic
molecules, on the other hand, cross the membrane
easily by diffusing through aqueous channels
formed by 'porin' molecules which span the membrane.
Loss of resistance to hydrophobic antimicrobial
substances occurs in 'deep rough' mutants and in
EDTA-treated cells [3,4] both of which have decreased amounts of protein a n d / o r LPS in the
outer layer of the outer membrane. Loss of protein
and LPS is compensated by an increase in phospholipid which then provides a pathway for the
inward diffusion of hydrophobic substances [10].
Exposure of phospholipid regions does not affect
resistance to small hydrophilic antibiotics entering
by the porin channels, whereas sensitivity to large
hydrophilic molecules e.g. vancomycin increases,
possibly due to transient ruptures forming in the
destabilised membrane [1].
Freeze-injured E. coli cells were sensitised to
hydrophobic but not to small hydrophilic antibiotics and thus resembled EDTA-treated cells or
those with deep rough mutations. The degree of
sensitisation to hydrophobic antibiotics varied:
sensitivity to small molecules generally increased
least. This may be because small hydrophobic
antibiotics can diffuse through porin channels to a
limited extent, hence any increase in sensitivity
due to damage would be less than with larger
molecules that were completely excluded by the
intact membrane.
Other damaging treatments produced generally
similar changes in the pattern of resistance though
the degree of sensitisation varied between treatments, possibly reflecting differences in the extent
of membrane disruption. Based on their relative
sensitivity to sodium deoxycholate, bacitracin,
novobiocin and vancomycin, frozen and thawed
cells had more severely disrupted membranes than
those heated or irradiated. Dried cells also had
severely damaged membranes but were unexpectedly resistant to novobiocin. This may be due
to a different form of membrane damage though it
is difficult to envisage structural changes which
would allow penetration of bacitracin whilst excluding the smaller, more hydrophobic, novobiocin.
Damaged cells all had increased cell surface
hydrophobicity, broadly reflecting their increased
sensitivity to hydrophobic antibiotics. Similar
changes have been reported in deep rough mutants
of Salmonella [11] which are also sensitive to hydrophobic antibiotics. The mechanisms causing
hydrophobicity changes in damaged cells are not
known. Loss or alteration of LPS has been reported in heated and frozen E. coli [12,13] and
membrane fragments shown to be released by
these treatments [14,15]. More detailed chemical
analysis of damaged membranes would help decide whether changes in hydrophobicity result from
loss of specific membrane components or from a
less specific disruption and reorganisation of the
membrane.
Despite probable differences in their modes of
action, the different stress treatments all resulted
in increased cell surface hydrophobicity which
probably explains their common effect of abolishing resistance to hydrophobic substances.
ACKNOWLEDGEMENTS
I would like to thank Mrs. J.A. Oxley and Miss
D.A. Cass for careful technical assistance.
399
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